Dr Marion BuckwalterMarion Buckwalter, MD, PhD
A search for biomarkers of functional and cognitive outcomes after a stroke has revealed a peripheral immune response occurring 2 days after an ischemic stroke that strongly predicts the likelihood of losing substantial mental acuity within 1 year.1

When regressed against the change in the Montreal Cognitive Assessment (MoCA) scores between day 90 and day 365 after the occurrence of stroke, the acute inflammatory phase Elastic Net (EN) model correlated with post-stroke cognitive trajectories (r = -0.692; Bonferroni-corrected P = .039). This, the authors indicated, suggest the helpfulness of immune profiling with mass cytometry to identify clinically relevant immune correlations with long-term cognitive trajectories.

Marion Buckwalter, MD, PhD, an associate professor of neurology and neurosurgery at Stanford School of Medicine, described the goal of their study. "Being able to identify, early on, patients who are at risk for dementia is a first step toward figuring out how to treat those at-risk patients," she said.

Buckwalter and colleagues, including co-senior authors of the study report Brice Gaudilliere, MD, PhD and Nima Aghaeepour, PhD, both Assistant Professors of Anesthesiology, Perioperative and Pain Medicine at Stanford, conducted a year-long longitudinal analysis of peripheral immune cell responses in 25 patients enrolled within 24 hours of stroke onset, and in a matched control cohort of healthy patients after hip replacement surgery.

The investigators note that previous studies of peripheral blood immune cells and secreted inflammatory mediators to find relatively accessible biological substrates of stroke mechanisms and recovery have predominantly focused on local and short-term (hours to days) immune responses. In addition, they find that previously available techniques that measured bulk cytokine response have provided insufficient insight into the biology that drives recovery.

In the current study, Buckwalter and colleagues applied mass cytometry to assess up to 50 phenotypic and functional parameters on a cell-by-cell basis. They combined this instrumentation with an unbiased EN method of analysis, with an algorithm refined in Aghaeepour's lab to hone complicated collections of highly correlated data, to comprehensively monitor the functional state of the immune system over a 1-year period.

"We didn't know what to expect," Aghaeepour acknowledged. "This was an exploratory study looking to understand how cells function after stroke. We had no idea we'd find a correlation with long-term cognitive outcomes. It was an exciting accident."

The correlation of immune system response and functioning to cognitive changes was able to be drawn with cognitive assessments of the participants taken on days 3, 30, 90 and 365. The principle cognitive measure was the MoCA, a 30-item cognitive screen. In addition, The Center for Epidemiological Studies-Depression (CES-D) and fatigue measure was administered, with family member assistance in responding if participants were affected by receptive aphasia. The short-form Neuro-QOL Fatigue Scale was also used to assess current levels of fatigue.

The investigators identified key elements of a robust and prolonged systemic immune response to ischemic stroke which were able to be differentiated into 3 distinct phases: an acute phase on day 2 after the stroke, characterized by increased signal transducer and activator of transcription 3 (STAT3) signaling responses in innate immune cell types; an intermediate phase (day 5) characterized by increased cAMP response element-binding protein (CREB) signaling responses in adaptive immune cell types; and a late phase (day 90) with persistent elevation of neutrophils and immunoglobulin M+ (Ig M+ ) B cells. By day 365, there was no detectable difference between these samples and those from the matched control cohort.

Buckwalter and colleagues reported finding that the distinct changes marking the acute phase, but not the intermediate or late phase, correlated with post-stroke cognitive decline, measured with MoCa scores, between day 90 and day 365 after stroke. The results were maintained after controlling for factors such as stroke location and size. The results demonstrate, they concluded, that deep immune profiling with mass cytometry can identify clinically relevant immune correlates of long-term cognitive trajectories.

"Our hypotheses is that when someone has a stroke, the molecules from the dead tissue are released and that when the immune system sees them and all is well, there is no immune response generated against them," Buckwalter explained to NeurologyLive.

"We think that the elevated immune responses just after the stroke act to over-stimulate the immune system during this time, producing a more harmful and long-lasting immune response," she elaborated. "We think these over-activated immune cells then migrate to the brain and attack the surviving tissue, like a slow simmering autoimmune response against the brain.".

Buckwalter and colleagues are currently conducting an expanded study with larger cohort which, she indicates, will more closely examine proteins in blood as well as stroke size and location, diet and medical history. Buckwalter hopes to better characterize the patients who are likely to manifest the acute phase immune response, and, perhaps, the means to mitigate it.

"Perhaps it is inherent/genetic, or it may be that it is just luck, or related to diet or exercise, or whether someone had recently had a cold," Buckwalter said. "We hope to learn more about this in our ongoing observational study."
REFERENCE
1. Tsai AS, Berry K, Benyto MM, et al. A year-long immune profile of the systemic response in acute stroke survivors. Brain. 2019.